Microorganism culture tests, which have been performed in order to, for example, determine a total aerobic count in food test, have conventionally comprised the following steps: dissolving agar powder for preparing a culture medium; sterilizing the medium; storing the medium at approximately 45° C. to avoid solidification; injecting a certain volume of the medium into a sterilized petri dish containing a certain volume of a sample solution (for example, a suspension of a food); solidifying the agar after mixing; culturing at constant temperature; and counting the number of colonies of microorganisms. This conventional method of microorganism culturing involves not only preparing a culture medium in advance and sterilizing the medium, but also storing the medium at a sufficiently high temperature to avoid solidification, thereby requiring much time and labor.
Demand exists for omitting these time-consuming preparation steps in order to conduct simple and rapid microorganism culture test. In addition, glass and plastic petri dishes used for the tests involve problems. In food tests, which require numerous tests of microorganism culturing, cleaning and sterilizing glass petri dishes for reuse are very time consuming. Disposable sterilized plastic petri dishes have recently come into wide used, raising another problem of handling of a large volume of plastic wastes. For the determination of total aerobic counts in an environmental microorganism test, a conventional method includes the steps of wiping a certain area of a test subject with a piece of gauze or cotton bud; rinsing the gauze or cotton bud in sterilized water or saline to thereby obtain a suspension of the microorganisms adsorbed on the gauze or cotton bud; applying the suspension on a prepared agar medium or mixing and diluting the suspension with an agar medium as described above; culturing the microorganisms; and counting microorganism colonies. This method is also very time- and labor-intensive.
In order to solve those problems, simplified culture media which do not require preparation processes have been studied, manufactured, and commercially distributed. These simplified culture media may be classified into 4 types: a press type, a filter type, a film type, and a test paper type.
Japanese Patent Application Laid-Open No. 4-117299 discloses an test method using a press-type medium, which comprises the steps of dispensing an agar medium into a plastic container having a mound-like shape; and bringing the medium into direct contact with a test subject. The method is simple and useful for testing microorganism pollution in an environment; however, it cannot provide quantitative results, because the medium has a small surface area, and difficult to employed to test subjects having a curved or uneven surface, thereby making the method inapplicable to conventional food tests. Like the conventional method, the method also involves a problem of generating a lot of plastic waste after culturing, because plastic containers are used.
ANSI/ASTM F 488-79 describes a test method using a filter-type medium, which is typically a water-absorbent substance containing nutrients and covered with a membrane filter, the method comprising immersing the culture medium in a sample solution for impregnation; catching microorganisms on the filter surface; and culturing the microorganisms. The filter-type medium is suitable for liquid samples, but encounters difficulty in test of solid-containing samples.
In a known test method using a test-paper-type medium, a filter paper is impregnated with nutrients for promoting growth of microorganisms, and a sample solution is added to or absorbed by the filter paper, which is then stored in a plastic bag for culturing. However, the paper medium cannot provide quantitative results, because cultured colonies are likely to disperse and microorganisms grown inside the paper are difficult to observe.
Japanese Patent Publication No. 2-49705 and Application Laid-Open No. 3-015379 disclose test methods employing film-type media. These methods use a laminate of two films sandwiching a nutrient and a gelling agent, which is reconstituted to a gel when cold water is added thereto. Subsequently, microorganisms are incubated by dispensing a sample solution between the films. The film-type medium provides quantitative results in food tests and hence is useful. In environmental monitoring tests, however, bringing the medium into direct contact with a subject is difficult, unlike the press-type medium, and nutrients or gelling agents that are not firmly bonded are likely to scatter. Other disadvantages include leaking of a solution due to degradation and liquification of the gel that occur when microorganisms that degrade the gelling agent grow in the medium and liquefy the gel.
As described above, several types of simplified culture media for specific purposes have been developed and commercially distributed. However, none of them have been applicable to multiple purposes, including food tests and direct test of an environment.
Japanese Patent Application Laid-Open No. 6-181741 discloses a microorganism culture device comprising a laminate of a cold water-soluble gelling agent, and a water-absorbent fiber sheet. With the combination of a gelling agent and a water-absorbing fiber sheet in the microorganism culture device, a portion of an added sample solution is absorbed by the gelling agent, thereby preventing uniform distribution of the sample solution on the surface of the culture device. In addition, the culture device involves a problem in obtaining quantitative results, because microorganisms may fail to form colonies on a top surface of the water-absorbing fiber sheet, and microorganisms grown on the lower surface of the fiber sheet or inside the sheet are difficult to count. Therefore, for practical use, this type of microorganism culture device requires a water-absorbing fiber sheet that is sufficiently thin to be transparent or semitransparent, thereby enabling easy observation of the grown microorganisms. Another disadvantage is that this culture device is not suitable to test methods involving pressing or direct wiping, because the water-absorbing fiber sheet and the layer of the cold water-soluble gelling agent are not bonded.
The present inventors have disclosed in International Patent WO 97/24432 a sheet- or film-form microorganism culture medium comprising a porous matrix layer and a water-soluble polymer layer. The culture medium is useful for various purposes, including normal food tests and tests by wiping, produces small wastes, and can provide quantitative results. In this type of medium, a sample solution added into the porous matrix layer is temporarily retained in the porous matrix layer. The water-soluble polymer layer coming in contact with the porous matrix layer dissolves in water in the retained sample solution, and simultaneously nutrients for microorganism growth contained in the water-soluble polymer layer also dissolve in the water, thereby initiating microorganism growth. When the sample solution is added to the medium, the microorganisms in the solution disperse in the porous matrix layer but not inside the water-soluble polymer layer. This is because a high-viscosity layer formed by gradual dissolution of the water-soluble polymer layer surface prevents movement of the microorganisms into the water-soluble polymer layer. The dissolved water-soluble polymer layer and the porous matrix layer are homogenized, thereby pressing the microorganisms up to the porous matrix layer surface, where colonies are formed. Therefore, counting of microorganisms is relatively easy despite use of the porous matrix layer, because colonies are formed on the porous matrix layer surface.
However, despite the excellent features of the sheet- or film-form culture container or medium comprising a porous matrix layer and a water-soluble polymer layer, some drawbacks are involved, due to lack of optimal physical properties of the porous matrix layer specifically for culturing microorganisms. For example, some sample solutions exhibit poor dispensability of microorganisms or a smaller number of observable microorganisms growing in the medium as compared with the case of a standard method. In addition, in the case where a significant number of microorganisms are present, growth of the microorganisms may be impossible to observe. Therefore, prior to culturing microorganisms, a user of this sheet- or film-form culture container or medium must consider various factors, including retention of the sample solution, its dispensability, or permeability of the matrix layer to the water-soluble polymer that is dissolved by the sample solution. Further, in microbial tests involving numerous samples, time-consuming bag-packing of the medium is required after sample addition, in order to prevent unwanted microorganisms from contaminating the medium, and the packed media are bulky.